Volume maintaining osteoinductive/osteoconductive compositions

ABSTRACT

An osteoinductive/osteoconductive composition prepared from a quantity, of demineralized fibrous bone elements possessing an average surface area to volume ratio of about 100:1 to about 20:1, a quantity of mostly shaped regular non-fibrous bone elements possessing an average surface area to volume ratio of about 10:1 or less and a sufficient quantity of biocompatible fluid carrier sufficient to provide the composition as a deformable mass is provided herein. Also provided is a method of using the composition to repair a bone defect site.

BACKGROUND OF THE INVENTION

[0001] This invention relates to an osteoinductive and osteoconductivecomposition containing demineralized fibrous bone elements incombination-with non-fibrous bone elements that are demineralized,partially demineralized or non-demineralized. More particularly, theinvention relates to demineralized fibrous bone elements having arelatively high median length to median thickness ratio and relativelyhigh surface area to volume ratio; demineralized, partiallydemineralized or non-demineralized non-fibrous bone elements that varyfrom “mostly irregular” to “mostly regular” in shape and not more than10 mm in any measurable component of the shape to determine size, e.g.,height, base, length, width, diameter or radius; and to a volumemaintaining osteoinductive/osteoconductive composition containing suchfibrous and non-fibrous elements within a biocompatible fluid carrier.

[0002] The use of pulverized exogenous bone growth material, e.g.,derived from demineralized allogenic or xenogenic bone in the surgicalrepair or reconstruction of defective or diseased bone is known. See, inthis regard, the disclosures of U.S. Pat. Nos. 4,394,370, 4,440,750,4,472,840, 4,485,097, 4,678,470, and 4,743,259; Bolander et al., “TheUse of Demineralized Bone Matrix in the Repair of Segmental Defects”,The Journal of Bone and Joint Surgery, Vol. 68-A, No. 8, pp. 1264-1273;Glowackie et al, “Demineralized Bone Implants”, Symposium on Horizons inPlastic Surgery, Vol. 12, No. 2; pp. 233-241 (1985); Gepstein et al.,“Bridging Large Defects in Bone by Demineralized Bone Matrix in the Formof a Powder”, The Journal of Bone and Joint Surgery, Vol. 69-A, No. 7,pp. 984-991 (1987); Mellonig, “Decalcified Freeze-Dried Bone Allograftas an Implant Material In Human Periodontal Defects”, The InternationalJournal of Periodontics and Restorative Dentistry, pp. 41-45 (June,1984); Kaban et al., “Treatment of Jaw Defects with Demineralized BoneImplants”, Journal of Oral and Maxillofacial Surgery, pp.623-626 (Jun.6, 1989); and, Todescan et al., “A Small Animal Model for InvestigatingEndosseous Dental Implants: Effect of Graft Materials on Healing ofEndosseous, Porous-Surfaced Implants Placed in a Fresh ExtractionSocket”, The International Journal of Oral & Maxillofacial Implants Vol.2, No. 4, pp. 217-223 (1987). According to Kakincki et al., “Human bonematrix gelatin as a clinical alloimplant”, International Orthopaedics,9, pp. 181-188 (1985), a water insoluble osteoinductivelosteoconductivesubstance referred to therein as “bone matrix gelatin” which wasobtained by decalcifying (ire., demineralizing) bone was successfullyemployed as an alloimplant for the treatment of bone defects and otherdisorders. An apparently similar water insolubleosteoinductive/osteoconductive material, referred to as “decalcifiedbone matrix”, is disclosed in McLaughlin et al., “Enhancement of BoneIngrowth by the Use of Bone Matrix as a Biologic Cement”, ClinicalOrthopaedics and Related Research, No. 183, pp. 255-261 (March, 1984).However, the prior art demineralized bone products have proven to beunsatisfactory for applications requiring a bone product, whichmaintains the volume of bone defect sites and allows for firm packing.Thus, an osteoinductive/osteoconductive composition, which maintains itscohesiveness and volume and resists erosion, would be highly desirable.

BRIEF SUMMARY OF THE INVENTION

[0003] It is an object of the invention to provide a quantity ofdemineralized fibrous bone elements, a quantity of non-fibrous boneelements that are demineralized, partially demineralized ornon-demineralized having a least dimension substantially larger than thethickness of the fibrous elements, and a cohesiveosteoinductive/osteoconductive composition containing the fibrous andnon-fibrous elements.

[0004] It is a further object of the invention to provide a cohesiveosteoinductive/osteoconductive composition, which is capable of wickingup blood and body fluids from the implant site, mixtures of bone marrowaspirate, autograft, etc.

[0005] It is a further object of the invention to provide the cohesiveosteoinductive/osteoconductive composition as an entangled mass with thenon-fibrous elements maintained within the entangled fibrous elements ofthe composition.

[0006] It is a further object of the invention to provide a cohesiveosteoinductive/osteoconductive composition with superior surgicalhandling properties, e.g., the ability to pick up globs of it withforceps in order to place it at a surgical site.

[0007] It is a further object of the invention to provide a volumemaintaining osteoinductive/osteoconductive composition, which can beplaced or injected into a hollow defect site.

[0008] It is a further object of the invention to provide anosteoinductive/osteoconductive composition having superior volumemaintaining properties, e.g., the ability to be packed firmly into largedefect sites.

[0009] It is a further object of the invention to provide anosteoinductive/osteoconductive composition that provides for rapidremodeling and incorporation of the non-fibrous elements into the hostsite, i.e., being turned into bone not only on the outside surfaces butalso on the internal surfaces of the non-fibrous elements such that thecomposition remodels from inside out as well as outside in.

[0010] It is a further object of the invention to provide a method oftreating trauma indications, e.g., tibia plateau fractures, such thatwhen the tibia plateau is elevated back to its normal anatomicalconfiguration, the crushed area of the metaphysis can be easily filledwith the composition of this invention to establish a solid fill thatcontributes to the maintenance of this normal anatomical reconstruction.

[0011] It is a further object of the invention to provide anosteoinductive/osteoconductive composition in which the size and shapeof the elements can be varied to suit the particular application.

[0012] It is a further object of the invention to provide anosteoinductive/osteoconductive composition in which the ratio of fibrouselements to non-fibrous elements to carrier can be varied to suit theparticular application.

[0013] It is a further object of the invention to provide a compositionthat is capable of being viewed utilizing radiographic imagingtechniques.

[0014] The stated objects of the invention are not intended to belimiting in any way. Of course further objects of the invention hereinwill be obvious to those skilled in the art in view of the above statedobjects and the foregoing specification.

[0015] In keeping with these and related objects of the invention, thereis provided an osteoinductive/osteoconductive composition comprising:(a) a quantity of fibrous bone elements possessing an average surfacearea to volume ratio of about 100:1 to about 20:1, (b) a quantity ofnon-fibrous bone elements possessing an average surface area to volumeratio of about 10:1 or less and (c) a quantity of biocompatible fluidcarrier sufficient to provide the composition as a deformable mass.Application of the foregoing osteoinductive/osteoconductive compositionto the site of a large bone defect, e.g., one resulting from injury,infection, disease, malignancy or developmental malformation, leads torapid new bone ingrowth by one or more mechanisms such as osteogenesis,osteoinduction and/or osteoconduction.

[0016] The inclusion of fibrous bone elements (a) imparts a higher levelof cohesiveness to the osteoinductive/osteoconductive composition ofthis invention compared with that of an osteoinductive/osteoconductivecomposition containing the same ratio of bone elements to carrier but inwhich the bone elements are all, or substantially all, of thenon-fibrous variety, e.g., the compositions of U.S. Pat. No. 5,073,373.The inclusion of non-fibrous bone elements (b) provides anosteoinductive/osteoconductive composition which requires a higher levelof applied mechanical force to effect its deformation than that requiredto deform an osteoinductive/osteoconductive composition containing thesame ratio of bone elements to carrier but in which all, orsubstantially all, of the bone elements possess a relatively highsurface area to volume ratio, e.g., the osteoinductive/osteoconductivecomposition of aforesaid U.S. Pat. No. 5,314,476 in which all, orsubstantially all, of the bone elements are of the fibrous variety.

[0017] The expression “median length to median thickness ratio” asapplied to the fibrous bone elements of the invention shall beunderstood to refer to the ratio of the longest median dimension of thefibrous bone element (its median length) to its shortest mediandimension (its median thickness).

[0018] The term “cohesive” as applied to theosteoinductive/osteoconductive composition of this invention shall beunderstood to refer to the ability of the composition to be shaped orpacked into a coherent mass which retains its shape and volume andresists erosion from the implant site.

[0019] The term “fibrous” as applied to this invention refers to boneelements whose median length to median thickness ratio is at least about10:1 and whose surface area to volume ratio is between about 100:1 andabout 20:1. In overall appearance the fibrous bone elements can bedescribed as fibers, threads, narrow strips, or thin sheets. Often,where thin sheets are produced, their edges tend to curl up toward eachother. The fibrous bone elements can be substantially linear inappearance or they can be coiled to resemble springs. The fibrous boneelements are preferably demineralized however some of the originalmineral content may be retained when desirable for a particularembodiment of the invention.

[0020] The expression “non-fibrous” as applied to the elements of thisinvention refers to elements that have a median width substantiallylarger than the median thickness of the fibrous bone element. Suchnon-fibrous bone elements will have a surface area to volume ratiosignificantly smaller than the fibrous bone elements, e.g., about 10:1or less. Preferably the non-fibrous bone elements are shaped in asubstantially regular manner or specific configuration, e.g. triangularprism, sphere, cube, cylinder and other regular shapes. By contrast,particles such as chips, shards, or powders possess irregular or randomgeometries. It should be understood that some variation in dimensionwill occur in the production of the elements of this invention andelements demonstrating such variability in dimension are within thescope of this invention and are intended to be understood herein asbeing within the boundaries established by the expressions “mostlyirregular” and “mostly regular”.

[0021] The expression “partially demineralized bone elements” as appliedto this invention refers to bone elements that are demineralized to theextent that only a small amount of mineral remains in the core. That isthe residual calcium is between about 50 to 100% by weight.

[0022] The expression “maintains its cohesiveness and volume and resistsdeformation” as applied to this invention refers to the ability of thecomposition to be packed into an appropriate size defect site and lockinto place remaining as a coherent mass where it is placed. In addition,the invention resists substantial deformation when subjected to a forceof up to about 10 N. This is in contrast to prior art compositions ofpaste-like or putty-like consistency as well as those of liquid or runnyconsistency.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following is a brief description of the drawings which arepresented only for the purposes of further illustrating the inventionand not for the purposes of limiting the same.

[0024]FIG. 1 are irregular non-fibrous bone elements, FIG. 1A are mostlyregular non-fibrous bone elements prepared as in Example 1. FIG. 1Brepresents a side-by-side comparison of the size and regular shape ofthe non-fibrous bone elements useful in the invention herein and theirregular non-fibrous elements of FIG. 1.

[0025]FIG. 2 represents the appearance of a prior art bone compositionand FIG. 2A is a composition prepared as in Example 1.

[0026]FIG. 3 is a relatively large defect site and FIG. 3A demonstratesthe ability of a composition prepared as in Example 1 to fill arelatively large defect site.

[0027]FIG. 4 represents the radiographic and biological results obtainedin Example 4.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The bone utilized in this invention is obtained utilizing methodswell known in the art, e.g., aseptically procured allogenic donor bonethat has been cleaned and disinfected. Fibrous bone elements whosemedian length to median thickness ratio is at least about 10:1 can bereadily obtained by any one of several methods, e.g., shaving thesurface of an entire bone or relatively large section of bone. Employinga shaving technique, fibrous bone elements ranging in median length fromabout 2 mm up to 400 mm or more (as in the case of the long bones) andin median thickness from about 0.05 mm to about 2 mm can be obtained. Anapparatus useful for obtaining the fibrous bone elements useful hereinis described in U.S. Pat. No. 5,607,269 the contents of which areincorporated herein by reference.

[0029] Depending on the procedure employed for producing the fibrousbone elements, one can obtain a mass of fibrous bone elements containingat least about 50 weight percent, preferably at least about 70 weightpercent and most preferably at least about 80 weight percent of thefibrous bone elements possessing a median length of from about 2 mm toabout 400 mm or more and preferably from about 10 mm to about 100 mm, amedian thickness of from about 0.05 mm to about 2 mm and preferably fromabout 0.08 mm to about 1.5 mm, and a median length to median thicknessratio of at least 10:1 up to about 500:1 or more and preferably fromabout 50:1 to about 100:1. The surface area to volume ratio of thefibrous bone elements will vary between about 100:1 and about 20:1,preferably between about 80:1 and about 40:1. If desired, the mass offibrous bone elements bone can be graded into different sizes and/or anyless desirable size(s) of fibrous bone elements which may be present canbe reduced or eliminated. The fibrous bone elements can be obtained fromcortical autogenic, cortical allogenic, cortical xenogenic, corticaltransgenic, cancellous autogenic, cancellous allogenic, cancellousxenogenic, cancellous transgenic, corticocancellous autogenic,corticocancellous allogenic, corticocancellous xenogenic orcorticocancellous transgenic bone. Porcine and bovine bone are aparticularly advantageous type of xenogenic bone tissue which can beused as a source for the fibrous bone elements of this invention,although of course other sources of bone such as ovine, caprine andequine may also be suitable.

[0030] Following shaving, milling or other technique whereby they areoptionally obtained, the fibrous bone elements are subjected todemineralization in order to reduce their inorganic content to a verylow level, e.g., to not more than about 5% by weight of residual calciumand preferably to not more than about 1% by weight residual calcium.Demineralization of the fibrous bone elements ordinarily results intheir contraction to some extent.

[0031] Demineralization of the fibrous bone elements can be conducted inaccordance with known conventional procedures. For example, in apreferred demineralization procedure, the fibrous bone elements aresubjected to an acid demineralization step that is followed by adefatting/disinfecting step. The bone is immersed in acid over time toeffect its demineralization. Acids which can be employed in this stepinclude inorganic acids such as hydrochloric acid and organic acids suchas peracetic acid. After acid treatment, the bone is rinsed with sterilewater for injection, buffered with a buffering agent to a finalpredetermined pH and then finally rinsed with water for injection toremove residual amounts of acid and buffering agent or washed with waterto remove residual acid and thereby raise the pH. Followingdemineralization, the bone is immersed in solution to effect itsdefatting. A preferred defatting/disinfectant solution is an aqueoussolution of ethanol, the ethanol being a good solvent for lipids and thewater being a good hydrophilic carrier to enable the solution topenetrate more deeply into the bone. The aqueous ethanol solution alsodisinfects the bone by killing vegetative microorganisms and viruses.Ordinarily at least about 10 to 40 weight percent by weight of water(i.e., about 60 to 90 weight percent of defatting agent such as alcohol)should be present in the defatting/disinfecting solution to produceoptimal lipid removal and disinfection within the shortest period oftime. The preferred concentration range of the defatting solution isfrom about 60 to 85 weight percent alcohol and most preferably about 70weight percent alcohol. Further in accordance with the invention, thedemineralized fibrous bone elements can be used immediately forpreparation of the osteoinductive/osteoconductive composition or theycan be stored under aseptic conditions, advantageously in a lyophilizedstate prior to such preparation. In a preferred embodiment, the fibrousbone elements can retain some of their original mineral content suchthat the composition is rendered capable of being imaged utilizingradiographic techniques such as disclosed in U.S. Pat. No. 5,676,146 thecontents of which are incorporated herein by reference.

[0032] The non-fibrous bone elements of this invention substantiallydisplay a relatively small surface area to volume ratio, e.g., less thanabout 10:1, preferably less than about 6:1, most preferably less thanabout 3:1. The median width of the non-fibrous bone elements is at leastas large as the median thickness of the fibrous bone elements utilizedin the composition of this invention but more preferably larger. Infurther accordance with some of the objects of this invention, the sizeand shape of the non-fibrous elements of the invention can be varied totailor the composition to its intended application. In preferredembodiments, the composition will contain non-fibrous elements thatexhibit a substantially larger least dimension than the least dimensionof the fibrous bone elements. In a preferred embodiment, the non-fibrousbone elements will display a “mostly regular” geometry, i.e.; the shapeof the non-fibrous bone elements is a triangular prism, sphere, cube,cylinder, other regular shape or a combination of these shapes. Suchshapes displaying a substantially regular geometry are to bedistinguished from chips, shards, and powders which may have arelatively small surface area to volume ratio but which due to their“mostly irregular” shape are unable to lock into place when used in acomposition intended to repair an appropriate size defect site. When itis desirable to have an embodiment capable of being injected or placedthrough, for example, a cannula or other similar device into a defectsite, the shape of the non-fibrous elements will be substantiallyspheroid. Such non-fibrous elements can be obtained from corticalautogenic, cortical allogenic, cortical xenogenic, cortical transgenic,cancellous autogenic, cancellous allogenic, cancellous xenogenic,cancellous transgenic, corticocancellous autogenic, corticocancellousallogenic, corticocancellous xenogenic or corticocancellous transgenicbone. Porcine and bovine bone are a particularly advantageous type ofxenogenic bone tissue which can be used as a source for the non-fibrousbone elements of this invention, although of course, ovine, caprine andequine bone may be entirely suitable.

[0033] About 20 to about 80 weight percent of the non-fibrous boneelements of the invention are non-fibrous bone elements having a medianlength to median width to median height ratio of at least about 1:0.3:1and up to about 1:1:5, a median length of from about 0.25 mm to about 10mm, a median width of from about 0.25 mm to about 10 mm and a medianheight of from about 0.25 mm to about 10 mm, the median width being thesmallest dimension of the non-fibrous element and the median heightbeing the io largest dimension of the non-fibrous element. Suchnon-fibrous elements are prepared utilizing methods well known in theart, e.g., cutting, milling, stamping, grinding. The size and shape ofthe non-fibrous elements can vary depending on the specific applicationthe composition is intended for, e.g., large trauma defects will requirerelatively large non-fibrous elements, whereas small dental defects,e.g., sinus lifts, three-wall defects, furcations, etc., will requirerelatively small non-fibrous elements. Such variation of size and shapeof the non-fibrous bone elements to tailor the composition to thespecific application is intended to be within the scope of thisinvention. The non-fibrous bone elements useful herein can be fullymineralized, partially demineralized, or fully demineralized (i.e., <5%calcium by weight). In a preferred embodiment of the invention, thecomposition contains from about 0 to about 50 percent by weight of thenon-fibrous bone elements mineralized bone, from about 0 to about 80percent by weight of the non-fibrous bone elements partiallydemineralized bone and from about 0 to about 100 percent by weight ofthe non-fibrous bone elements fully demineralized bone.

[0034] The bone utilized in making the non-fibrous elements of theinvention can be fully mineralized, partially demineralized or fullydemineralized prior to the preparation of the non-fibrous elements. In apreferred embodiment cortical bone is cut into slices, e.g., about 3 mmin width, and then demineralized to the extent that only a small amountof mineral remains in the core, i.e., less than 10% by weight residualcalcium, preferably less than 5% by weight residual calcium. The bone isthen cut with a stamping technique to yield substantially cuboid shapesabout 3×3×3 mm in length, width and height. Optionally, mineralized boneis cut into substantially cuboid shapes with a band saw. The bone cubesare then demineralized using techniques well known in the art, e.g.,such as those described above. After the non-fibrous elements areobtained they can be used immediately for preparation of theosteoinductive/osteoconductive composition or they can be stored underaseptic conditions, advantageously in a lyophilized or frozen stateprior to such preparation.

[0035] To prepare an osteoinductive/osteoconductive compositionutilizing the demineralized fibrous bone elements and non-fibrous boneelements of this invention, a quantity of the fibrous and non-fibrouselements are combined with an amount of biocompatible fluid carrierwhich will provide a coherent mass. The carrier can be any of a numberof compounds and/or polymers, e.g., polymer sugars, proteins, long chainhydrophilic block copolymers, reverse phase block copolymers, hyaluronicacid, polyuronic acid, mucopolysaccharide, proteoglycan,polyoxyethylene, surfactants, e.g., the pluronics series of nonionicsurfactants, and peptide thickener. Suggested classes of biocompatiblefluid carrier would include polyhydroxy compound, polyhydroxy ester,fatty alcohol, fatty alcohol ester, fatty acid, fatty acid ester, liquidsilicone, mixtures thereof, and the like.

[0036] Examples of suitable biocompatible fluid carrier include, but arenot limited to:

[0037] (i) Polyhydroxy compound, for example, such classes of compoundsas the acyclic polyhydric alcohols, non-reducing sugars, sugar alcohols,sugar acids, monosaccarides, disaccharides, water-soluble or waterdispersible oligosaccarides, polysaccarides and known derivatives of theforegoing. Specific polyhydroxy compounds include,1,2-propanediol,glycerol, 1,4,-butylene glycol trimethylolethane, trimethylolpropane,erythritol, pentaerythritol, ethylene glycols, diethylene glycol,triethylene glycol, tetraethylene glycol, propylene glycol, dipropyleneglycol; polyoxyethylene-polyoxypropylene copolymer, e.g., of the typeknown and commercially available under the trade names Pluronic andEmkalyx; polyoxyethylene-polyoxypropylene block copolymer, e.g., of thetype known and commercially available under the trade name Poloxamer;alkylphenolhydroxypolyoxyethylene, e.g., of the type known andcommercially available under the trade name Triton, polyoxyalkyleneglycols such as the polyethylene glycols, xylitol, sorbitol, mannitol,dulcitol, arabinose, xylose, ribose, adonitol, arabitol, inositol,fructose, galactose, glucose, mannose, sorbose, sucrose, maltose,lactose, maltitol, lactitol, stachyose, maltopentaose,cyclomaltohexaose, carrageenan, agar, dextran, alginic acid, guar gum,gum tragacanth, locust bean gum, gum arabic, xanthan gum, amylose,mixtures of any of the foregoing, and the like.

[0038] (ii) Polyhydroxy ester, for example, liquid and solid monoestersand diesters of glycerol can be used to good effect, the solid estersbeing dissolved up to the limit of their solubilities in a suitablevehicle, e.g., propylene glycol, glycerol, polyethylene glycol of200-1000 molecular weight, etc. Liquid glycerol esters include monacetinand diacetin and solid glycerol esters include such fatty acidmonoesters of glycerol as glycerol monolaurate, glyceryl monopalmitate,glyceryl monostearate, etc. An especially preferred carrier hereincomprises glyceryl monolaurate dissolved in glycerol or a 4:1 to 1:4weight mixture of glycerol and propylene glycol, poly (oxyalkylene)glycol ester, and the like.

[0039] (iii) Fatty alcohol, for example primary alcohols, usuallystraight chain having from 6 to 13 carbon atoms, including caproicalcohol, caprylic alcohol, undecyl alcohol, lauryl alcohol, andtridecanol.

[0040] (iv) Fatty alcohol ester, for example, ethyl hexyl palmitate,isodecyl neopentate, octadodecyl benzoate, diethyl hexyl maleate, andthe like.

[0041] (v) Fatty acid having from 6 to 11 carbon atoms, for example,hexanoic acid, heptanoic acid, octanoic acid, decanoic acid andundecanoic acid.

[0042] (vi) Fatty acid ester, for example,polyoxyethylene-sorbitan-fatty acid esters; e.g., mono- and tri-lauryl,palmityl, stearyl, and oleyl esters; e.g., of the type available underthe trade name Tween from Imperial Chemical Industries; polyoxyethylenefatty acid esters; e.g., polyoxyethylene stearic acid esters of the typeknown and commercially available under the trade name Myrj; propyleneglycol mono- and di-fatty acid esters such as propylene glycoldicaprylate; propylene glycol dilaurate, propylene glycol hydroxystearate, propylene glycol isostearate, propylene glycol laureate,propylene glycol ricinoleate, propylene glycol stearate, and propyleneglycol caprylic-capric acid diester available under the trade nameMiglyol; mono-, di-, and mono/di-glycerides, such as the esterificationproducts of caprylic or caproic acid with glycerol; e.g., of the typeknown and commercially available under the trade name lmwitor; sorbitanfatty acid esters, e.g., of the type known and commercially availableunder the trade name Span, including sorbitan-monolauryl,-monopalmityl,-monostearyl, -tristearyl, -monooleyl and triolcylesters;monoglycerides, e.g., glycerol mono oleate, glycerol mono palmitate andglycerol monostearate, for example as known and commercially availableunder the trade names Myvatex, Myvaplex and Myverol, and acetylated,e.g., mono- and di-acetylated monoglycerides, for example, as known andcommercially available under the trade name Myvacet; isobutyl tallowate,n-butylstearate, n-butyl oleate, and n-propyl oleate.

[0043] (vii) Liquid silicone, for example, polyalkyl siloxanes such aspolymethyl siloxane and poly (dimethyl siloxane) and polyalkylarylsiloxane.

[0044] In a preferred embodiment of the osteoinductive/osteoconductivecomposition, the liquid carrier is a liquid polyhydroxy compound, liquidpolyhydroxy compound derivative, liquid solution of solid polyhydroxycompound, liquid solution of solid polyhydroxy compound derivative ormixtures thereof. If necessary or desirable, the liquid carrier can bedissolved or diluted with an appropriate solvent such that when combinedwith the fibrous and non-fibrous elements of the invention a compositioncapable of being shaped or packed into a coherent mass which retains itsshape and volume over the relatively long term, e.g., until the boneformation and remodeling process is completed, is provided. Thus, thepolyhydroxy compound or polyhydroxy derivatives can be a liquid in thepure or highly concentrated state at ambient temperature, e.g., 1.5 -50°C., or it can be a solid or semi-solid at this temperature in which caseit becomes necessary to dissolve the material in a solvent such aswater, physiological saline, ethanol, glycerol, glucose, propyleneglycol, polyethylene glycol of from 200-1000 molecular weight, polyvinylalcohol, etc. Of course, the liquid carrier can be made up of one ormore liquid polyhydroxy compounds or derivatives in solution with one ormore solid polyhdroxy compounds or derivatives.

[0045] Of the foregoing polyhydroxy compounds, glycerol and its liquidmonesters and diesters, e.g. monacetin and diacetin, fructose, glucoseand sucrose, and mixtures thereof are preferred. Where the polyhydroxycompound is a solid, e.g., sucrose, a solvent such as water, glycerol,polyethylene glycol of from 200-1000 average molecular weight, ormixture thereof is used to provide a cohesive solution or paste of thecompound.

[0046] Where, in a particular osteoinductive/osteoconductivecomposition, the fibrous and/or non-fibrous elements exhibit a tendencyto quickly or prematurely separate from the carrier component or tootherwise settle out from the composition such that application of afairly homogeneous composition is rendered difficult or inconvenient, itcan be advantageous to include within the composition an optionalsubstance whose thixotropic characteristics prevent or reduce thistendency. Thus, e.g., where the carrier component is glycerol andseparation of fibrous and/or non-fibrous bone elements occurs to anexcessive extent where a particular application is concerned, athixotropic agent such as a solution of polyvinyl alcohol,polyvinylpyrrolidone, cellulosic ester such as hydroxypropylmethylcellulose, carboxyl methylcellulose, pectin, food-gradetexturizing agent, gelatin, dextran, collagen, starch, hydrolyzedpolyacrylonitrile, hydrolyzed polyacrylamide, polyelectrolyte such aspolyacrylic acid salt, hydrogels, chitosan, other materials that cansuspend particles, etc., can be combined with the carrier in an amountsufficient to significantly improve the suspension-keepingcharacteristics of the composition.

[0047] If desired, the fibrous and/or non-fibrous bone elements of thisinvention can be modified in one or more ways, e.g., their proteincontent can be augmented or modified as described in U.S. Pat. Nos.4,743,259 and 4,902,296 the contents of which are incorporated herein byreference. Any of a variety of medically and/or surgically usefuloptional substances can be incorporated in, or associated with, the boneelements before, during, or after preparation of theosteoinductive/osteoconductive composition. Thus, e.g., one or more ofsuch substances can be introduced into the bone elements, e.g., bysoaking or immersing the bone elements in a solution or dispersion ofthe desired substance(s), by adding the substance(s) to the carriercomponent of the osteoinductive/osteoconductive composition or by addingthe substance(s) directly to the osteoinductive/osteoconductivecomposition.

[0048] Medically/surgically useful substances which can be readilycombined with the bone elements, fluid carrier and/orosteoinductive/osteoconductive composition of this invention include,e.g., demineralized bone powder as described in U.S. Pat. No. 5,073,373the contents of which are incorporated herein by reference, collagen,insoluble collagen derivatives, hydroxyapatite, etc., and soluble solidsand/or liquids dissolved therein, e.g., antiviricides, particularlythose effective against HIV and hepatitis; antimicrobials and/orantibiotics such as erythromycin, bacitracin, neomycin, penicillin,polymyxin B, tetracyclines, viomycin, chloromycetin and streptomycins,cefazolin, ampicillin, azactam, tobramycin, clindamycin and gentainycin;etc.; amino acids, peptides, vitamins, inorganic elements, inorganiccompounds, cofactors for protein synthesis, hormones; endocrine tissueor tissue fragments; synthesizers; enzymes such as collagenase,peptidases, oxidases, etc.; polymer cell scaffolds with paraenchymalcells; angiogenic drugs and polymeric carriers containing such drugs;collagen lattices; biocompatible surface active agents; antigenicagents; cytoskeletal agents; cartilage fragments, living cells such aschondrocytes, bone marrow cells, mesenchymal stem cells, naturalextracts, tissue transplants, bioadhesives, bone morphogenic proteins(BMPs), transforming growth factor (TGF-beta), insulin-like growthfactor (IGF-1) (IGF-2), platelet derived growth factor (PDGF),fibroblast growth factors (FGF), vascular endothelial growth factor(VEGF), angiogenic agents, bone promoters, cytokines, interleukins,genetic material, genes encoding bone promoting action, cells containinggenes encoding bone promoting action; growth hornones such assomatotropin; bone digestors; antitumor agents; fibronectin; cellularattractants and attachment agents; immuno-suppressants; permeationenhancers, e.g., fatty acid esters such as laureate, myristate andstearate monesters of polyethylene glycol, surface active agents,enamine derivatives, α-keto aldehydes, etc.; nucleic acids; epidermalgrowth factor (EGF); all collagen types (not just type 1);non-collagenous proteins such as osteopontin, osteonectine, bone sialoproteins, vitronectin, thrombospondin, proteoglycans, decorin, biglycan,aggrecan, versican, tenascin, matrix gla protein hyaluronan; soluble andinsoluble components of the immune system, soluble and insolublereceptors including truncated forms, soluble, insoluble and cell surfacebound ligands including truncated forms; chemokines, bioactive compoundsthat are endocytosed; compounds capable of altering the membranepotential of cells, compounds capable of altering the monovalent anddivalent cation/anion channels of cells; bone resportion inhibitors andstimulators; angiogenic and mitogenic factors; bioactive factors thatinhibit and stimulate second messenger molecules; integrin adhesionmolecules; clotting factors; externally expanded autograft or xenograftcells and any combinations thereof. The amounts of such optionally addedsubstances can vary widely with optimum levels being readily determinedin a specific case by routine experimentation.

[0049] As previously indicated, the osteoinductive/osteoconductivecomposition of this invention can be freshly prepared just by mixingdesired quantities of the demineralized fibrous bone elements,non-fibrous bone elements, fluid carrier and optional component(s), ifany, in any suitable sequence of separate mixing, adsorption,rehydration or drying operations or all at once. Thus, the demineralizedfibrous bone elements and/or non-fibrous bone elements can be mixed withthe optional ingredients(s) and thereafter combined with the fluidcarrier component, the demineralized fibrous bone elements and/ornon-fibrous bone elements can be mixed with the fluid carrier followedby addition of the optional ingredient(s) or the optional ingredientscan be added to the fluid carrier followed by addition of thedemineralized fibrous bone elements and/or non-fibrous bone elements.Variations of these and other sequences of mixing are, of course,possible. Advantageously, the fibrous and non-fibrous elements and fluidcarrier are mixed substantially simultaneously such that the fibrouselements of the osteoinductive/osteoconductive composition are entangledand the non-fibrous bone elements are thoroughly mixed in the entangledfibrous bone elements.

[0050] The amount of demineralized fibrous bone elements which can.beincorporated into the osteoinductive/osteoconductive composition canvary widely with amounts of from about 5 to about 90 weight percent, andpreferably from about 20 to about 70 weight percent, being entirelysuitable in most cases. Likewise, the amount of the non-fibrous boneelements which can be incorporated into theosteoinductive/osteoconductive composition can very widely with amountsfrom about 10 to about 90 weight percent, and preferably from about 20to about 70 weight percent, being entirely suitable in most cases. Theratio of fibrous to non-fibrous bone elements can vary between about0.2:1 to about 1:0.2. The balance of the composition being made up offluid carrier and optional ingredient(s), if any.

[0051] In embodiments where it is desirable to improve the ability ofthe osteoinductive/osteoconductive composition to be imaged, e.g., byx-ray, radiopaque material(s) may be incorporated into the composition.Such materials would include, e.g., barium sulfate, iodine-containingcompounds, titanium and mineralized bone.

[0052] To facilitate on-site preparation and/or usage of the compositionherein, the demineralized fibrous bone elements and non-fibrous boneelements, preferably in lyophilized or frozen form, and fluid carrier(the latter containing one or more optional ingredients such as thoseidentified above) can be stored in separate packages or containers understerile conditions and brought together in intimate admixture at themoment of use for immediate application to an osseous defect siteemploying any suitable means such as spatula, forceps, syringe, tampingdevice, etc. Alternatively, the osteoinductive/osteoconductivecomposition can be prepared well in advance and stored under sterileconditions until required for use. When theosteoinductive/osteoconductive composition is prepared well in advanceit is preferably lyophilized prior to packaging for storage. At the timejust prior to when the osteoinductive/osteoconductive composition of theinvention is to be placed in a defect site optional materials, e.g.,autograft bone marrow aspirate, autograft bone, preparations of selectedautograft cells, autograft cells containing genes encoding bonepromoting action, etc., can be combined with the composition of thisinvention. Preferably, the osteoinductivelosteoconductive composition ispackaged already mixed and ready for use in a suitable container, e.g.,syringe, resealable non-toxic bottle, etc., or is provided as a kitwhich can be prepared at a surgeon's direction when needed.

[0053] The osteoinductive/osteoconductive composition of this inventioncan be firmly placed into an appropriate size defect site to maintainvolume and provide support for adjacent tissues. Such placement can beaccomplished through the use of a variety of devices such as, e.g.,spatula, forceps, syringe, tamping device, etc.

[0054] The osteoinductive/osteoconductive composition of this inventioncan be tailored to be utilized for a variety of orthopaedic,neurosurgical, and oral and maxillofacial surgical indications in whichit would be advantageous to be able to firmly place the composition intoa bone defect site such as the repair of simple and compound fracturesand nonunions, external fixations, joint reconstructions such asarthrodesis, general arthroplasty, acetabular repair, cup arthroplastyof the hip, femoral and humeral head replacement, femoral head surfacereplacement and total joint replacements, repairs of the vertebralcolumn including spinal fusion and internal fixation, tumor surgery,e.g., deficit filling, discectomy, lain inectomy, excision of spinalcord tumors, anterior cervical and thoracic operations, repair of spinalinjuries, scoliosis, lordosis and kyphosis treatments, intermaxillaryfixation of fractures, mentoplasty, temporomandibular joint replacement,alveolar ridge augmentation and reconstruction, inlay bone grafts,implant placement and revision, sinus lifts, furcation defects,periodontal defects, dental defects, ulna defects, metaphyseal defects,tibia plateau defects, wrist defects, ankle defects, etc.

[0055] The invention will be more fully understood by way of thefollowing examples which are intended to illustrate but not limitmethods of preparation of the demineralized fibrous bone elements andnon-fibrous bone elements of the invention and the preparation of anosteoinductive/osteoconductive composition containing the fibrous andnon-fibrous elements in accordance with the present invention. Acomparison of the compressive force of prior art compositions and thecomposition of the invention is also provided, however, this comparisonis intended to illustrate but not limit the differences between thisinvention and the prior art.

EXAMPLE 1

[0056] Sections of defatted, disinfected allogenic cortical boneapproximately 210-250 mm in length were cut on a band saw to yield145.65 g of cuboid non-fibrous bone elements about 3 mm in size. Theremaining allogenic cortical bone was processed in the bone millingapparatus described in U.S. Pat. No. 5,607,269 to yield 145.8 grams offibrous bone elements. The non-fibrous bone elements were then placed ina reactor. A 0.6 N solution of HCl at 15 ml per gram of non-fibrous boneelements was introduced into the reactor, the reaction proceeded for 1to 2 hours. Following drainage of the HCl, the non-fibrous bone elementswere covered with 0.6 N HCl/20 ppm-2000 ppm nonionic surfactant solutionfor 24 to 48 hours. The fibrous bone elements were then added to thereactor and allowed to soak for 5 to 10 minutes. Following drainage ofthe HCl/surfactant solution, 0.6 N HCl at 15 ml per gram of total bonewas introduced into the reactor, the reaction proceeded for 40 to 50minutes. Following drainage through a sieve the bone was rinsed threetimes with water for injection at 15 ml per gram non-fibrous elementweight with the water for injection being replaced at 15-minuteintervals. Following drainage of the water for injection, the bone wascovered with alcohol and allowed to soak for at least 30 minutes. Thealcohol was then drained and the bone was rinsed with water forinjection. The bone was then contacted with a mixture of 3.5 ml ofglycerol per gram of dry bone and 5 ml of water for injection per gramof dry bone for at least 2 hours. After draining, the composition wastransferred to a lyophilization tray and frozen at −70° C. for at least6 hours. The composition was then lyophilized following standardprocedures for 24 to 48 hours.

EXAMPLE 2

[0057] The compressive force of the composition prepared as in Example 1was compared with that of a like quantity of anosteoinductive/osteoconductive composition prepared in accordance withU.S. Pat. No. 5,073,373 and an osteoinductive/osteoconductivecomposition prepared in accordance with U.S. Pat. No. 5,314,476. In thisexample, 5 cc of each material was placed into separate 10 cc syringebarrels. The compressive force (i.e., the sustained force capable ofdeflecting a meter probe) was then measured using the meter, todetermine deflective force. The results are contained in the followingtable. Material Compressive Force (N) U.S. Patent No. 5,073,373 4.8 U.S.Patent No. 5,314,476 7.9 Example 1 10.3

EXAMPLE 3

[0058] Material prepared as in Example 1 was evaluated to determine itsosteoinductive potential. The material was implanted in female athymichomozygous rnu/rnu rats according to the procedure described in Edwardset al., Osteoinduction of Human Demineralized Bone: Characterization ina Rat Model, Clinical Orthopedics and Related Research (No. 357, pp.219-228) 1998, the contents of which are incorporated hereby byreference. The material was studied to analyze its bone formationresponse. After 28 days in the rat model it was determined that cellshad accumulated in the porous region inside of the chips, differentiatedinto bone forming cells, and were in the process of laying down bone inremodeling the matrix from the inside out.

EXAMPLE 4

[0059] Twenty-four 6 month-old (3.5-4.0 kg) male New Zealand whiterabbits (Covance: Denver, Pa.) were used. The animals received astandard rabbit diet (Purina, Ind.) and received standard tap water adlibitum. The animals were kept on a 12 h light/dark cycle.

[0060] The animals underwent surgery to create bilaterial 1.5 cm ulnardefects by the method described by Bostrom et al., Use of Bonemorphogenic Protein-2 in the Rabbit Ulnar Nonunion Modelz, ClinicalOrthopedics and Related Research (No. 327, pp. 272-282) 1996, thecontents of which are incorporated herein by reference. The 48 defectswere randomly assigned and implanted with one of the four graftingmaterials (Table 1). The final volume of each implant was 1 cc. Theanimals were not restricted from full weight bearing after surgery.TABLE 1 EVAULATION TREATMENT GROUP PERIODS SAMPLE SIZE 1 cc ofautogenous bone 6 and 12 weeks N = 1 at 6 weeks graft from the iliaccrest N = 9 at 12 weeks Demineralized fibers with 6 and 12 weeks N = 3at 6 weeks demineralized cortical N = 8 at 12 weeks chips Demineralizedfibers with 6 and 12 weeks N = 3 at 6 weeks non-demineralized cortical N= 8 at 12 weeks chips DBM Putty with non- 6 and 12 weeks N = 3 at 6weeks demineralized cancellous N = 9 at 12 weeks chips Empty 12 weeks N= 2 at 12 weeks

[0061] The animals were sedated and serial radiographs of the forelimbswere taken every three weeks until 12 weeks post-operatively when theanimals were sacrificed. At the time of sacrifice, the ulnas wereremoved, cleaned of soft tissue, and radiographed using a highresolution Faxitron. Bony union (Table 2) and quantitative boneformation (Table 3) was evaluated at each time point by 3 independent,blinded observers. Bony union was defined as bridging of the defect inexcess of 25% of the diaphyseal diameter. The radiographs were digitizedto normalize the bone area and intensity so that bone formation could bequantified using image analysis software. Bone formation was evaluatedon a standardized 5-point scale measuring percent of new bone seen indefect: 0=no new bone evident in defect, 1=1-25%, 2=26-50%, 3=51-75%,4=76-99%, 5=100%. TABLE 2 RADIOGRAPHIC UNIONS BY TIME POINT 9 Weeks 12Weeks TREATMENT GROUP Union Nonunion Union Nonunion Autograft 5 4 8 1Demin Fibers/Demin 1 7 7 1 Cortical Chips DBM Putty/Non-Demin 2 6 7 1C/C Chips Demin Fibers/Non- 0 9 6 3 Demin Cortical Chips

[0062] TABLE 3 BONE FORMATION EVALUATED RADIOGRAPHICALLY AREA OF DEFECTOCCUPIED BY BONE (MEDIAN SCORE) TREATMENT GROUP Week 3 Week 6 Week 9Week 12 Autograft 0 4 4 5 Demin Fibers/Demin 0 3 3.5 4 Cortical ChipsDBM Putty/Non-Demin 1 3 4 4 C/C chips Demin Fibers/Non-Demin 1 3 3 4Cortical Chips

[0063] All limbs from each group were prepared for histologicalanalysis. Tissue samples were dehydrated over a course of several weekswith daily changes of the alcohol solutions. After dehydration wascomplete, tissue samples were embedded in methylmethacrylate. The blockswere cut in the longitudinal direction of the bone in 5 μm sectionsusing a microtome. Serial sections were stained with one of thefollowing stains: Hematoxylin and eosin, Goldner-Masson trichrome, orVon Kossa. The sections were examined for cellular characteristicsindicative of new bone formation and callus formation. Groups using theFisher's exact test compared radiographic union data. Bone formation wasverified with a nonparametic analysis of variance (ANOVA), theKruskal-Wallis H-test.

[0064] Experimental protocols were followed without incidence. There wasone postoperative death resulting in the loss of 2 experimental defects.One defect was lost from demineralized fibers and demineralized corticalchips. Radiographic evaluation showed no statistically significantdifferences between the four groups. Some bone formation was evident inall groups by six weeks. At 12 weeks, all groups displayed similarquantities of new bone formation as assessed by radiodensity scale.(Table 3).

[0065] Union of the defect sites occurred in a similar fashion in allgroups (Table 2). The autogenous bone graft (“ABG”) group did showhigher union rates at earlier point times, but at 12 weeks there is nostatistically significant difference between the three groups. Strongbony bridges were seen in all four groups at 12 weeks. The onlyexceptions were in the demineralized fibers with non-demineralized chip.In this group, though 6 out of 9 were united, the defects tended not tohave maintained their three-dimensional space and showed sagging in themiddle.

[0066] It shall be understood, however, that the scope of the presentinvention is not to be limited to the specific embodiments describedabove. The invention may be practiced other than as particularlydescribed and still be within the scope of the accompanying claims.

What is claimed is:
 1. An osteoinductive/osteoconductive compositioncomprising: (a) a quantity of demineralized fibrous bone elementspossessing an average surface area to volume ratio of about 100:1 toabout 20:1, (b) a quantity of shaped non-fibrous bone elementspossessing an average surface area to volume ratio of about 10:1 or lessand, (c) a sufficient quantity of biocompatible fluid carrier sufficientto provide the composition as a deformable mass.
 2. Theosteoinductive/osteoconductive composition of claim 1 of which about 50to about 100 percent by weight of the fibrous bone element is made up ofdemineralized fibrous bone elements having a median length of from about2 mm to about 400 mm, a median thickness of from about 0.05 mm to about2 mm and a median length to median thickness ratio of at least 10:1 upto about 500:1.
 3. The osteoinductive/osteoconductive composition ofclaim 1 in which a shape of the non-fibrous bone elements is selectedfrom the group consisting of triangular prism, sphere, cube, cylinderand other regular shapes.
 4. The osteoinductive/osteoconductivecomposition of claim 1 wherein the bone elements are obtained fromcortical autogenic, cortical allogenic, cortical xenogenic cancellousautogenic, cancellous allogenic, cancellous xenogenic, corticaltransgenic, cancellous transgenic, corticocancellous autogenic,corticocancellous allogenic, corticocancellous xenogenic orcorticocancelldus transgenic bone.
 5. The osteoinductive/osteoconductivecomposition of claim 1 of which about 20 to about 80 weight percent ofthe non-fibrous bone elements of the invention are non-fibrous boneelements having a median length to median width to median height ratioof at least about 1:0.3:1 and up to about 1:5:1, a imedian length offrom about 1 mm to about 10 mm, a median width of from about 1 mm toabout 10 mm and a median height of from about 1 mm to about 10 mm. 6.The osteoinductive/osteoconductive composition of claim 1 wherein about0 to about 50 percent by weight of the bone elements are mineralized. 7.The osteoinductive/osteoconductive composition of claim 1 wherein about0 to about 80 percent by weight of the bone elements are partiallydemineralized.
 8. The osteoinductive/osteoconductive composition ofclaim 1 wherein about 0 to about 100 percent by weight of the boneelements are demineralized.
 9. The osteoinductive/osteoconductivecomposition of claim 1 further comprising a thixotropic agent.
 10. Theosteoinductive/osteoconductive composition of claim 1 further comprisingat least one medically/surgically useful substance.
 11. Theosteoinductive/osteoconductive composition of claim 1 wherein thefibrous bone elements are entangled.
 12. Theosteoinductive/osteoconductive composition of claim 11 wherein thenon-fibrous bone elements are thoroughly mixed in the entangled fibrousbone elements.
 13. The osteoinductive/osteoconductive composition ofclaim 1 containing from about 20 to about 70 weight percentdemineralized fibrous bone elements, from about 20 to about 70 weightpercent non-fibrous bone elements, and from about 10 to about 80 weightpercent fluid carrier.
 14. The osteoinductive/osteoconductivecomposition of claim 1 wherein the ratio of fibrous to non-fibrouselements is about 0.2:1 to about 1:0.2.
 15. Theosteoinductive/osteoconductive composition of claim 1 further comprisingat least one additive selected from the group consisting of autograftbone marrow aspirate, autograft bone, preparations of selected autograftcells, autograft cells containing genes encoding bone promoting actionand autograft cells expanded outside the body and returned.
 16. Theosteoinductive/osteoconductive composition of claim 1 wherein thecomposition withstands a force of at least about 7.9 N withoutsignificant deformation.
 17. The osteoinductive/osteoconductivecomposition of claim 1 wherein the composition withstands a force of atleast about 10.3 N without significant deformation.
 18. Theosteoinductive/osteoconductive composition of claim 1 wherein the amountof mineral remaining in the elements allows for radiographic imaging ofthe composition.
 19. The osteoinductive/osteoconductive composition ofclaim 1 further comprising at least one radiopaque material selectedfrom the group consisting of barium sulfate, iodine containingcompounds, titanium and mineralized bone.
 20. A method of using theosteoinductive/osteoconductive composition of claim 1 wherein thecomposition is packed appropriately into an appropriate size bone defectsite.
 21. The method of claim 20 wherein the composition is packed intothe defect site utilizing at least one means selected from the groupconsisting of spatula, forceps, syringe and dental equipment.
 22. Themethod of claim 20 wherein the defect site is selected from the groupconsisting of ulna defects, metaphyseal defects, tibia plateau defects,acetabular defects, sinus defects, long bone cortical defects, cranialdefects, ilium defects, wrist/hand defects, ankle/foot defects andoral/maxillofacial defects.
 23. The method of claim 20 wherein thecomposition further comprises at least one additional additive selectedfrom the group consisting of autograft bone marrow aspirate, autograftbone, autograft cell preparations, autograft cells containing genesencoded for bone stimulating activity and autograft cells expandedoutside the body and returned.
 24. The osteoinductive composition ofclaim 1 containing about X weight units of component (a), about Y weightunits of component (b) and about Z weight units of component (c) a givenamount of the composition exhibiting a greater resistance to deformationthan the identical amount of a second osteoinductive/osteoconductivecomposition containing X+Y weight units of component (a), no amount ofcomponent (b) and Z weight units of component (c).